7. Project Summary/Abstract Our goal is to understand how the HIV-1 5?-Leader RNA directs multiple, diverse functions during viral replication, including selective packaging of the dimeric, unspliced viral genome during virus assembly. Significant progress made during the current funding period includes: (1) We determined the 3D structure of the HIV-1 packaging signal, a longstanding milestone. (2) We showed that transcriptional start site heterogeneity affords RNA transcripts that contain one, two, or three 5?-guanosines and exhibit different structures, functions, and fates; these findings established a new paradigm for controlling genome versus mRNA fates. (3) We discovered that the intact 5?-leader rapidly adopts an extended dimer interface, even in the absence of RNA chaperones. (4) We showed that packaging elements reside primarily in the 5?-leader and do not extend into downstream regions of the gag gene, as previously proposed. (5) Several NMR methods were developed that extend the size limit for RNA structural studies, including (i) a 2H-edited NMR/mutagenesis approach for measuring RNA dimerization dynamics, (ii) a hybrid NMR/cryoEM approach applicable to relatively small RNAs, including the HIV dimer initiation site, and (iii) a method that extends the RNA size limit for residual dipolar coupling (RDC) measurement by more than an order of magnitude compared to existing approaches. In addition, we now have preliminary evidence that transcriptional start site heterogeneity modulates splicing, in addition to translation and packaging. We are now poised to determine the 5?-leader structures of the unspliced and spliced HIV RNAs generated by heterogeneous transcriptional start site usage. These studies will explain how transcriptional inclusion of as few as one or two 5?-guanosines has such a profound influence on RNA structure and function. Preliminary studies also reveal that truncated HIV Gag protein constructs (comprising the capsid through the nucleocapsid domains of Gag) bind the cognate, dimeric RNA packaging signal with high affinity and ~18:2 Gag:RNA stoichiometry. We are now also poised to determine the 3D structure of this complex by hybrid NMR/EM, which will provide the first molecular views of the Gag:RNA complex that nucleates virus assembly. NMR studies of large RNAs are technically challenging ? the average size of NMR-derived RNA structures in the RNA Structure Database is only 27 nucleotides ? but the potential payoff is substantial and could ultimately lead not only to a more detailed understanding of how HIV replicates, but also to the development of new approaches for the treatment of AIDS and other virally-induced human diseases.